The mammalian branched-chain alpha-keto acid (BCKA) dehydrogenase complex catalyzes oxidative decrboxylation of alpha-keto acids derived from the branch-chain amino acids (BCAA), leucine, isoleucine and valine. The enzyme complex consists of three catalytic components; a decarboxylase (E1b), a dihydrolipoyl trnasacylase (E2b) and a dihydrolipoyl dehydrogenase (E3); and two regulatory enzymes: a kinase and a phosphatase. The proposed work is aimed to elucidate structure and function of the catalytic components of the mammalian BCKA dehydrogenase complex. Our approaches will be at three different levels. (1) Protein chemistry: We have developed sensitive assays for the overall reaction and for each catalytic component of the BCNA dehydrogenase complex. E1b and E2b components have been isolated from the highly purified complex of bovine liver, and initially characterized. We will further study subunit structure of these two components by limited proteolyusis, HPLC, amino acid sequence analysis and physical methods. Our aim is to establish reaction and control mechanisms of the BCKA dehydrogenase complex, and to investigate possible structural homologies between transacylase (E2b) and transsuccinylase (E2b) subunits. (2) mRNA studies: We have recently obtained highly specific antisera E2b, E1b, -alpha and E1b -beta subunits of the bovine BCKA dehydrogenase complex. Specific mRNA encoding each polypeptide precursor of the complex will be prepared from 3T3- 1.1 adipocytes or bovine liver by polysome immunoadsorption. These mRNA's will be used as templates to study synthesis and import into mitochondiria of the above protein subunits using the cell-free translation system. (3) Molecular cloning: The enriched mRNA will be a source for cloning of cDNA for subunits of the complex in gtil or phBK322 library. The antiserum against SDS- denatured E2b that recognizes a broad spectrum of antigenic site will be a useful probe for isolation of E2b -encoding cDNA cloies from the gtll expression library. E. coli gene clones will also be utilized as possible probes to screen E2b gene sequences form a bovine cDNA library. The molecular cloning will allow future investigation of the enzyme complex at the level of the genes. Results obtained from these studies may provide insights into the evolution and biogenesis of the mammalian BCKA dehydrogenase complex. The information will be relevant to understanding the role of BCAA and BCNA oxidation in diabetes, liver cirrhosis nad the heritable maple-syrup-urine disease.